We want to purify our proteins. After dialyzing the protein we eluted them by concentration gradiant of NaCl in Ion exchange column. We need to know concentration of our fractions. For this, we used of Bradford method but proteins precipitate after reagent adding.
As i think there is two main reason for precipitation during bradford . as we know one reason is existence of detergents as bradford is much more sensitive for detergents but in my opnion in your work the second reason may occur which is delaying for much more time. may be the sample was extended for much time and dye dye aggregates formed or may be the sample is not well mixed with the bradford reagent.
We need to know that other than NaCl which buffer system and what detergent you have used to elute your protein from the Ion exchange column, as they may intefere in your Bradford assay method. In case you have used certain detergents try using RC DC protein assay kit available with Biorad which is compatible to a variety of Reducing agents and Detergents... Wishing you good luck... best regards
Dear Deepti
We used phosphate buffer for dialyze in exchanging of NaCl from our samples. We didnt use any detergent in this experiment.
Frankly speaking bradford assay is compatible with 5M NaCl so it should not interfere in your assay. Even bradford is compatible with 0.1M Phosphate buffer.. So if your concentration is not exceeding these limits its pretty good. In this case then we need to understand why your protein is precipitating. Try using Lowry and BCA as well and if the same happens in these assay methods also, then probably your protein is denaturated or has lost its original configuration...
Hi Mohammad,
i had similar problems by using a fractionation protocol. Bradford is not compatible with different reagents, such as SDS, HEPES and so on. I suggest you to check the compatibility of your buffer, and of course the limits of reagent concentration tolerated by Bradford assay (please see the attached file). In any case, i solved this problem by using the Lowry method
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